A particular challenge in the delivery of a gene by a viral vector or a virus for therapeutic purposes is the preparation and accurate quantification of clinical dosage forms. The production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to the ever changing demands and applications.
Virus titer determination or virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. Traditional methods include viral plaque assays which determine the number of plaque forming units (pfu) in a virus sample and the Tissue Culture Infective Dose (TCID50) or Fluorescence Active Infectious Dose (FAID50) which measures the infectious virus titer. This TCID50 assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells. The traditional methods are generally slow and labor-intensive, and suffer from limitations including a high degree of inter-assay variability.
Enzyme-Linked Immunosorbent Assay (ELISA) is a more modern variation of a protein assay that utilizes a specific antibody linked to an enzyme to detect the presence of an unknown amount of antigen (i.e. virus) in a sample. The antibody-antigen binding event is detected and/or quantified through the enzyme's ability to convert a reagent to a detectable signal that can be used to calculate the concentration of the antigen in the sample. The plate assays of the virus titer determination that are based on immunofluorescence detection using an ELISA are developed, however they are used to quantify proteins from virus samples and not to quantify infectious viruses.
Flow cytometry or FACS (fluorescence-activated cell sorter) assays have been used to measure the number of infected cells in cell cultures infected at relatively high multiplicities of infection. For example, U.S. Pat. No. 6,248,514 discloses the use of flow cytometry to analyze cells infected using specified ranges of viral particle concentration and adsorption time yields. U.S. Pat. No. 7,476,507 discloses FACS-based methods for the determination of the viral titer of a culture of host animal host cells infected with a circovirus. However, for many applications, the cost, size and complexity of the flow cytometry instruments prevent wider use.
Recently, personal image cytometers are developed which combine the functions of a digital microscope, an image cytometer, and a cell counter in a single benchtop instrument. The commercially available image cytometers include CellInsight (ThermoFisher) and Cytell Cell Imaging System (GE Healthcare). These image cytometers are used to make individual cell, subcellular and multi-cellular measurements, and provide the opportunity for scientists to capture cellular and subcellular image data and analyze results with ease.
The instant invention addresses the need for an accurate and high throughput method of quantitating infectious viral particles in a sample.